KR20170028187A - Method for controlling polyhydroxyalkanoate using sequence variation in 5'-untranslated region - Google Patents

Abstract

The present invention relates to a method for producing a polyhydroxyalkanoate by culturing cells comprising polyhydroxyalkanoate (PHA) synthase where 5-untranslated region sequence is controlled and, more specifically, to a method for controlling a polyhydroxyalkanoate content by controlling a 5-untranslated region sequence of polyhydroxyalkanoate synthase.

Description

[0001] The present invention relates to a method for controlling polyhydroxyalkanoate content in a 5'-untranslated region,

There is provided a method for producing a polyhydroxyalkanoate by culturing a cell comprising a polyhydroxyalkanoate (PHA) synthase whose 5'-untranslated region sequence is regulated do. Also provided is a method for regulating the intracellular polyhydroxyalkanoate content by controlling the 5'-untranslated region sequence of a polyhydroxyalkanoate synthase.

Polyhydroxyalkanoate (PHA) is a microorganism that accumulates in microorganisms for the storage of energy and reducing ability when a microorganism is abundant in a carbon source in a state in which an element necessary for growth of nitrogen, oxygen, phosphorus, It is a natural polyester material. PHA has been recognized as a substitute for conventional synthetic plastics because it exhibits similar biodegradability and biocompatibility with synthetic polymer derived from petroleum.

Most of the known monomers are PHA monomers such as 3-, 4-, 5- or 6-hydroxyalkanoate (HA), and PHA monomers which are actively studied 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), 3-hydroxypropionate (3HP), and an intermediate chain having 6 to 14 carbon atoms Monomers having a hydroxyl group at carbon positions 3 and 4, such as 3-hydroxyalkanoate of medium chain length (MCL), and the like.

Conventionally, in order to increase the content of PHA polymer in microorganisms, various heterologous enzyme genes involved in the synthesis of PHA in microorganisms are introduced, or enzyme mutant genes with modified amino acid sequence are introduced to change its activity, The method of changing the content has been mainly used.

However, such conventional techniques are difficult to precisely control intracellular polymer content desired by the experimenter, and screening for a large number of mutants has to be performed in order to achieve desired contents.

Korean Patent Publication No. 2008-0047279, May 28, 2008 Korean Patent Publication No. 2013-0059308, June 5, 2013

The present invention relates to a method for regulating the sequence of a 5'-untranslated region (5'-UTR) involved in the post-transcription level during the expression of intracellular proteins, the present invention relates to a technology capable of precisely controlling the expression amount of a polyhydroxyalkanoate (PHA) synthase, and ultimately achieving the intracellular polyhydroxyalkanoate content only by the control of the 5'-non-translated region.

One example provides a method for producing a polyhydroxyalkanoate, comprising culturing a cell comprising a polyhydroxyalkanoate synthase with a 5'-untranslated region sequence regulated.

In one embodiment, the method comprises coupling a regulated 5'-untranslated region with a PHA synthase gene, and introducing a PHA synthase gene linked to the regulated 5'-non-translated region into the cell. A method for controlling the content of polyhydroxyalkanoate in a cell is provided.

In another embodiment, the 5'-untranslated region comprises the nucleotide sequence of 5'-NNNNNNNNNN-AAAGGAGCATC-NNNN-3 'or 5'-NNNNNNN-CAGAGAGAC-NNNNNNNNN-3' G or < / RTI > C.

In another embodiment, the 5'-untranslated region is a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: .

In another embodiment, the PHA synthase gene may be a PHA synthase gene of Pseudomonas sp. 6-19.

In another embodiment, the PHA synthase gene comprises the amino acid sequence of SEQ ID NO: 29; Or a nucleotide sequence corresponding to an amino acid sequence comprising at least one mutation selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: .

In another embodiment, the polyhydroxyalkanoate synthase gene comprises (i) S325T and Q481M in the amino acid sequence of SEQ ID NO: 29; (ii) E130D, S325T and Q481M; (iii) E130D, S325T, S477R and Q481M; And (iv) a mutation selected from the group consisting of E130D, S477F and Q481K.

In another embodiment, the cell may be a prokaryotic cell.

Another example provides a method of modulating intracellular polyhydroxyalkanoate content, comprising the step of regulating the 5'-untranslated region sequence of a PHA synthase.

In one embodiment, the method comprises coupling a regulated 5'-untranslated region with a PHA synthase gene, and introducing a PHA synthase gene linked to the regulated 5'-non-translated region into the cell. A method for controlling the content of polyhydroxyalkanoate in a cell is provided.

In another embodiment, the 5'-untranslated region comprises the nucleotide sequence of 5'-NNNNNNNNNN-AAAGGAGCATC-NNNN-3 'or 5'-NNNNNNN-CAGAGAGAC-NNNNNNNNN-3' G or < / RTI > C.

In another embodiment, the 5'-untranslated region may comprise a nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 25.

In another embodiment, the PHA synthase gene may be a PHA synthase gene of Pseudomonas sp. 6-19.

In another embodiment, the PHA synthase gene comprises the amino acid sequence of SEQ ID NO: 29; Or a nucleotide sequence corresponding to an amino acid sequence comprising at least one mutation selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: .

In another embodiment, the polyhydroxyalkanoate synthase gene comprises (i) S325T and Q481M in the amino acid sequence of SEQ ID NO: 29; (ii) E130D, S325T and Q481M; (iii) E130D, S325T, S477R and Q481M; And (iv) a mutation selected from the group consisting of E130D, S477F and Q481K.

In another embodiment, the cell may be a prokaryotic cell.

The present invention is based on the fact that the expression level of polyhydroxyalkanoate synthase can be finely controlled by controlling the 5'-nontranslational region sequence involved in the post-transcriptional step during intracellular protein expression, It was confirmed that the content of polyhydroxyalkanoate in the cells had a linear relationship. Thus, ultimately, the inventors have found that the intracellular polyhydroxyalkanoate polymer content desired can be achieved only by the regulation of the 5'-untranslated region.

Accordingly, in one aspect, the present invention provides a technique for controlling the intracellular polyhydroxyalkanoate content by controlling the 5'-untranslated region sequence of the PHA synthase.

In another aspect, the present invention provides a technique for producing polyhydroxyalkanoate with high efficiency by introducing a PHA synthase gene having a maximized expression amount through a custom design of a 5'-untranslated region sequence into cells to provide.

The 5'-untranslated region refers to a region that exists at the 5'-end of the mRNA transcript but is not translated into an amino acid. The process by which the mRNA is translated into a protein begins with the 30S subunit of the ribosome binding to the 5'-untranslated region. Specifically, 16S rRNA (16S ribosomal RNA) in the 30S subunit of the ribosome binds to the ribosome binding site (RBS) in the 5'-untranslated region and tRNA recognizes the initiation codon (AUG) of the mRNA When combined, translation into protein begins. The ribosome binding site and the initiation codon of the 5'-untranslated region are approximately 6 to 8 nucleotides apart, and there is a complementary sequence in the ribosome binding site complementary to the 3 'terminal sequence of 16S rRNA, Shine-Dalgarno) sequence.

Accordingly, in a preferred embodiment, the 5'-untranslated region of the present invention includes a sequence complementary to the 3'-terminal sequence of 16S rRNA, that is, a Shine-Dalgano sequence, .

Herein, the term "5'-untranslated region sequence-regulated" or "regulated 5'-non-translation region" refers to a modification of one or more bases in the base sequence constituting the 5'-untranslated region, Deformation includes substitution, addition, deletion or inversion of the base. As a representative example, the Shine-Dalgarno sequence in the 5'-untranslated region is conserved, and one or more bases in the nucleotide sequences existing before and after the 5'-ungranslated region may include a sequence modified by substitution, addition, deletion, or inversion .

For the regulation of the 5'-untranslated region sequence, a 5'-untranslated region design program known in the art can be used. For example, UTR Designer (Seo et al., Metabolic Engineering 15 (2013) 67-74) can be used.

For example, in order to increase the expression level of the PHA synthase gene and ultimately increase the intracellular polyhydroxyalkanoate content, the 5'-untranslated region sequence has a free binding energy? G _UTR value Can be adjusted to a sequence having a value smaller than the ΔG _UTR value of the original sequence (wild-type sequence), and conversely, in order to decrease the expression amount of the PHA synthase gene and ultimately decrease the intracellular polyhydroxyalkanoate content the 5'-untranslated region sequences can be defined free binding energy ΔG _UTR sequence having a value adjusted to a value greater than ΔG _UTR of the original sequence (wild type sequence), as follows. In the above,? G _UTR means the difference in Gibbs free energy before and after binding of the 30S subunit complex of the ribosome to the mRNA transcript, and can be expressed by the following equation:

ΔG _UTR = [ΔG _SD + ΔG _start + ΔG _spacing ] - [αΔG _direct + (1 -?)? G _indirect ]

Where G _SD is the energy released when the Shine-Dalgarno sequence hybridizes to 16S rRNA,

ΔG _start is the energy released when the initiation codon hybridizes with the initiator tRNA (3'-UAC-5 '),

ΔG _spacing is the free energy penalty of an unoptimized physical distance between the Shine-Dalgarno sequence and the initiation codon,

ΔG _direct Is the energy required for the 30S subunit of the ribosome to bind directly to the translation initiation site when the translation initiation site of the transcript is in a temporarily unfolded state,

ΔG _indirect is the energy required for the 30S subunit of the ribosome to bind to the translation initiation site by an indirect force rather than a direct linkage when the translational initiation site of the transcript is in an unfolded state,

α is the population coefficient.

In the above, the values of ΔG can be calculated using NUPACK (Dirks R. M. et al., SIAM Rev. 49 (2007) 65-88).

In one embodiment, the 5'-untranslated region may comprise the nucleotide sequence of 5'-NNNNNNNNNN-AAAGGAGCATC-NNNN-3 'or 5'-NNNNNNN-CAGAGAGAC-NNNNNNNNN-3'. In this case, N means A, T, G or C.

In a specific example of the present invention, a 5'-untranslated region sequence variant prepared by changing the 25 bases (-25 to -1) before the start sequence of the PHA synthase using the ReC promoter was used, Enzyme activity and cell polyhydroxyalkanoate contents were investigated. According to this, it was confirmed that the expression amount of PHA synthase was regulated by controlling the 5'-non-translated region sequence, and the amount of enzyme expression and thus the content of polyhydroxyalkanoate in the cells had a linear relationship.

For example, the 5'-untranslated region sequences regulated to increase the expression level of the PHA synthase gene and ultimately increase the intracellular polyhydroxyalkanoate content include SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 25.

In contrast, the 5'-untranslated region sequences regulated in order to reduce the expression level of the PHA synthase gene and ultimately reduce the intracellular polyhydroxyalkanoate content are shown in SEQ ID NO: 1 to SEQ ID NO: 12, SEQ ID NO: 15, A nucleotide sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24 can be exemplified. However, this is merely an example for understanding the present invention, and the scope of the present invention is not limited thereto.

In the examples of the present invention, in the case of SEQ ID NO: 19 and SEQ ID NO: 22, the amount of the polyhydroxyalkanoate in the cells was somewhat decreased as compared with the amount of the enzyme expressed in the original UTR sequence. Therefore, it can be utilized to improve the polymer content by using the UTR mutation sequence in another enzyme expression system (or other strain) in which the expression level of the enzyme is low and the polymer content is low. Thus, the technical significance of the present invention is not limited only to the specific strains provided in the examples or the 5'-untranslated region sequence variants of the specific sequence numbers, but can be utilized in various enzyme expression systems or various strains to produce intracellular polymers / RTI > and / or < / RTI >

In addition, although the present example provides an example in which the amount of PHA synthase and the amount of polyhydroxyalkanoate in the cell are regulated by controlling the 5'-untranslated region sequence, this is merely a typical application example of the present invention, It is clear that the present invention can be applied to the expression of various enzymes and the production of various polymers in cells in the future. For example, in the course of developing a strain producing a desired polymer, it is possible to simultaneously express various proteins by controlling the 5'-untranslated region sequence using the present invention. However, excessive expression of a large number of proteins may act as intracellular stress and excess energy may be used for protein synthesis. As a result, the desired polymer may not be produced smoothly. Therefore, the UTR sequence in which the content of the polymer Can be selected for polymer production.

In the present invention, the polyhydroxyalkanoate (PHA) synthesizing enzyme may be a PHA synthase gene of Pseudomonas sp. 6-19.

In one embodiment, the polyhydroxyalkanoate synthase gene is

An amino acid sequence of SEQ ID NO: 29; or

A nucleotide sequence corresponding to an amino acid sequence comprising at least one mutation selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: have.

In another embodiment, the polyhydroxyalkanoate synthase gene comprises the amino acid sequence of SEQ ID NO: 29,

(i) S325T and Q481M;

(ii) E130D, S325T and Q481M;

(iii) E130D, S325T, S477R and Q481M; And

(iv) a nucleotide sequence corresponding to an amino acid sequence comprising a mutation selected from the group consisting of E130D, S477F and Q481K.

In addition, the gene encoding the enzyme may comprise a nucleic acid molecule comprising a functionally equivalent codon or a codon encoding the same amino acid (by codon degeneracy), or a codon encoding a biologically equivalent amino acid have. The nucleic acid molecule may be isolated or prepared using standard molecular biology techniques, for example, a chemical synthesis method or a recombinant method, or a commercially available nucleic acid molecule may be used.

In the present invention, specific examples for adjusting the 5'-untranslated region to ultimately control the polyhydroxyalkanoate or improve its production include,

Linking the regulated 5'-untranslated region with a PHA synthase gene, and

And introducing the PHA synthase gene linked to the regulated 5'-non-translated region into the cell.

As described above, the modulation of the 5'-untranslated region can be performed using the UTR Designer or by adjusting the sequence of the 5'-untranslated region so that the defined free binding energy? _UTR value is changed.

The 5'-untranslated region thus regulated can be introduced into the cell in conjunction with the PHA synthase gene. For example, a regulated 5'-untranslated region and a PHA synthase gene can be cloned into a single vector and introduced into the cell. Herein, the vector refers to a gene construct comprising an essential regulatory element operably linked to the expression of a gene insert encoding a desired protein in a cell of an individual, and may be in various forms such as a plasmid, a viral vector, a bacteriophage vector, Can be used.

A vector inserted into a vector and fused to the genome of a host cell irreversibly to allow the gene expression to remain stable for a long period of time is preferable. Such vectors include transcriptional and detoxification expression control sequences that allow the gene to be expressed in a selected host. Expression control sequences may include promoters for conducting transcription, any operator sequences for regulating such transcription, and / or sequences that control termination of transcription and translation. The initiation codon and the termination codon are generally considered to be part of the nucleic acid sequence encoding the target protein and should act in the individual when the gene construct is administered and in the coding sequence and in frame. The promoter of the vector may be constitutive or inducible. It may also contain a cloning source if it is a replicable expression vector. In addition, an enhancer, an untranslated region at the 3 'end of the desired gene, a selectable marker (for example, an antibiotic resistance marker), or a replicable unit may suitably be contained. The vector may be self-replicating or integrated into the host genomic DNA.

Each component in the vector must be operatively linked to each other and the linkage of these component sequences can be performed by ligation at a convenient restriction site and if such a site is not present, For example, using a synthetic oligonucleotide adapter or a linker.

Examples of useful expression control sequences include early and late promoters of adenovirus, monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, long terminal repeat (LTR) promoter of HIV, Moloney virus, (CMV) promoter, Epstein virus (EBV) promoter, Roussacomavirus (RSV) promoter, RNA polymerase II promoter, T3 and T7 promoters, major operator and promoter regions of phage lambda, and prokaryotic or eukaryotic Or other sequences of constitutions and derivatives known to modulate the expression of the genes of their viruses, and various combinations thereof.

Introduction into the cell means that the PHA synthase gene linked to the regulated 5'-non-translated region is introduced into the host cell so that the nucleic acid can be replicated as an extrachromosomal element or by chromosomal integration. The cell may be a prokaryotic cell, e.g., E. coli (e.g., E. coli DH5a, E. coli JM101, E. coli K12, E. coli W3110, E.coli X1776, E. coli B, and E . coli XL1-Blue), Escherichia spp., Pseudomonas spp., Bacillus spp., Streptomyces spp., Erwinia spp., Serratia spp., Providencia spp., Corynebacterium spp., Leptospira spp., Salmonella spp. Bacterium, hypomonas, chromobacterium, norcadia, and the like. Once introduced into a suitable host cell, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself.

For the purpose of the present invention, the host cell may be a cell having a pathway for biosynthesizing a polyhydroxyalkanoate from a carbon source using a PHA synthase, or may be a cell having a pathway from a carbon source to a polyhydroxyalkanoate May be a cell engineered to be able to biosynthesize.

Introduction of the PHA synthase gene linked to the regulated 5'-untranslated region into the host cell can be carried out using suitable standard techniques, such as electroporation, electroinjection, Microinjection, calcium phosphate co-precipitation, calcium chloride / rubidium chloride method, retroviral infection, DEAE-dextran, cationic liposomes, a liposome method, a polyethylene glycol-mediated uptake method, a gene gun, and the like, but the present invention is not limited thereto. At this time, the vector of circular form can be introduced into a linear vector form by cutting with appropriate restriction enzymes.

The medium used for cell culture should suitably meet the requirements of a particular strain. The medium may include various carbon sources, nitrogen sources, phosphorus, and trace element components. Carbon sources in the medium include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil and coconut oil, palmitic acid, Fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Examples of the nitrogen source in the medium include peptone, yeast extract, juice, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, But is not limited thereto. The nitrogen source may also be used individually or as a mixture. Examples of the materials in the medium include, but are not limited to, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. In addition, the culture medium may include metal salts such as magnesium sulfate or iron sulfate necessary for growth, or may include essential growth materials such as amino acids and vitamins, but is not limited thereto. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

In addition, if necessary, the pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid in a suitable manner. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. Oxygen or oxygen-containing gas (e.g., air) may be injected into the culture to maintain aerobic conditions, and the temperature of the culture may be usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The culture can continue until the desired yield of the desired terpolymer is obtained.

The polyhydroxyalkanoate produced in the present invention may contain a hydroxyalkanoate monomer as a repeating unit or a repeating unit of two or more hydroxyalkanoate monomers.

For example, the hydroxyalkanoate monomer may be 2-, 3-, 4-, 5- or 6-hydroalkenoate. As specific examples, there may be mentioned 2-hydroxybutyrate, glycolate, lactate, 3-hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate (3- hydroxyvalerate, a medium chain length (MCL) MCL 3-hydroxyalkanoate having 6 to 14 carbon atoms (for example, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydrononanoate, 3-hydroxydecanoate, 3-hydroxydecanoate, 3-hydroxyundecanoate and 3-hydroxydodecanoate), 4-hydroxybutyrate, 4-hydroxyvalerate, and the like. , 4-hydroxyhexanoate, 4-hydroxyheptanoate, Hydroxyheptanoate, 4-hydroxyoctanoate, 4-hydrononanoate, 4-hydroxydecanoate, 5-hydroxy- 5-hydroxyvalerate, 5-hydroxyhexanoate, 6-hydroxydodecanoate, 3-hydroxy-4-pentenoic acid, Hydroxy-4-trans-hexenoic acid, 3-hydroxy-4-cis-hexenoic acid, 3-hydroxy- Hexenoic acid, 3-hydroxy-6-trans-octenoic acid, 3-hydroxy-6-cis-octenoic acid, Octenoic acid, 3-hydroxy-8-nonenoic acid, 3-hydroxy-9-decenoic acid, 3-hydroxy hydroxy-5-cis-dodecenoic acid, 3-hydroxy-6-cis-dodecene Dodecenoic acid, 3-hydroxy-5-cis-tetradecenoic acid, 3-hydroxy-7-cis-tetradecenoic acid, (hydroxy) -5,8-cis-cis-tetradecenoic acid, 3-hydroxy-4-methylvaleric acid, 3-hydroxy- Methylhexanoic acid, 3-hydroxy-5-methylhexanoic acid, 3-hydroxy-6-methylheptanoic acid, 3-hydroxy- 4-methyloctanoic acid, 3-hydroxy-5-methyloctanoic acid, 3-hydroxy-6-methyloctanoic acid, 3- hydroxy-7-methyloctanoic acid, 3-hydroxy-6-methylnonanoic acid, 3-hydroxy-7-methylnonanoic acid, 3-hydroxy-8-methylnonanoic acid, 3- Hydroxy-7- methyldecanoic acid, hydroxy-9-methyldecanoic acid, 3-hydroxy-7-methyl-6-octenoic acid ( octenoic acid, malic acid, 3-hydroxysuccinic acid-methyl ester, 3-hydroxyadipinic acid-methyl ester, 3-hydroxysuberic acid-methyl Ester, 3-hydroxyazelaic acid-methyl ester, 3-hydroxysebacic acid-methyl ester, 3-hydroxysuberic acid-ethyl ester, Hydroxysebacic acid-ethyl ester, 3-hydroxypimelic acid-propyl ester, 3-hydroxysebacic acid-benzyl ester, 3-hydroxy-8-acetic acid Acetoxyoctanoic acid, 3-hydroxy-9-acetoxynonanoic acid, Phenoxy-3-hydroxybutyric acid, phenoxy-3-hydroxyvaleric acid, phenoxy-3-hydroxyheptanoic acid, Phenoxy-3-hydroxyoctanoic acid, cyanophenoxy-3-hydroxybutyric acid, cyanophenoxy-3-hydroxynaphthoic acid, Hydroxyvaleric acid, para-cyanophenoxy-3-hydroxyhexanoic acid, para-nitrophenoxy-3-hydroxyhexanoic acid, 3 5-phenylvaleric acid, 3-hydroxy-5-cyclohexylbutyric acid, 3,12-dihydroxydodecanoic acid, Dihydroxy-5-cis-tetradecenoic acid, 3-hydroxy-4,5-epoxydecanoic acid oic acid, 3-hydroxy-6,7-epoxydodecanoic acid, 3-hydroxy-8,9-epoxy-5,6-cis- Hydroxybenzoic acid, tetradecanoic acid, cyano-3-hydroxyheptanoic acid, 9-cyano-3-hydroxynonanoic acid, 3-hydroxy Fluoroheptanoic acid, 3-hydroxy-9-fluorononanoic acid, 3-hydroxy-6-chlorohexanoic acid, 3-hydroxy-8-chloroooctanoic acid, 3-hydroxy-6-bromohexanoic acid, 3-hydroxy- bromooctanoic acid, 3-hydroxy-11-bromoundecanoic acid, 3-hydroxy-butenoic acid, 6-hydroxy- Dodecenoic acid, 3-hydroxy-2-methylbutyrate (methylbutyri one selected from the group consisting of acetic acid, 3-hydroxy-2-methylvaleric acid and 3-hydroxy-2,6-dimethyl-5-heptenoic acid. Or more.

According to the present invention, intracellular polyhydroxyalkanoate contents can be finely controlled through the regulation of the 5'-untranslated region.

Figure 1 graphically shows the linear correlation of relative expression (%) of polyhydroxyalkanoate synthase with intracellular polyhydroxyalkanoate content (wt%) according to the 5'-UTR sequence regulation .
Fig. 2 shows a cleavage map of the vector pPs619C1249.18H-CPPCT540.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. 5'- UTR  Sequence-regulated pHA  Preparation of transformants containing synthetic enzymes

1-1. The controlled 5'- UTR  Generation of sequence

(5'-GTGACGG CAGAGAGAC AATCAAATC-3 ', SEQ ID NO: 26) using the UTR Designer (Seo et al., Metabolic Engineering 15 (2013) 67-74) (5'-NNNNNNN-CAGAGAGAC-NNNNNNNNNN-3 ') to generate the regulated 5'-UTR sequence of SEQ ID NO: 1 to SEQ ID NO: 19, preserving the Shine-Dalkano sequence and altering the other parts 5'-NNNNNNNNNN-AAAGGAGCATC-NNNN-3 ') generated the regulated 5'-UTR sequence of SEQ ID NO: 20 to SEQ ID NO: 25.

1-2. pPs619C1249 .18H- CPPCT540  Manufacture of vector

As a PHA synthesizing enzyme using lactyl-CoA and 4-hydroxyalkanoyl-CoA as a substrate, a mutant of a PHA synthase derived from Pseudomonas sp. MBEL 6-19 (KCTC 11027BP) was used. In order to remove the PHA synthase (phaC1 _{Ps6 -19)} gene, the total DNA extracted of Pseudomonas species MBEL 6-19 (KCTC 11027BP) and, based on the phaC1 _{Ps6 -19} gene sequence (SEQ ID NO: 30), primer 5 (SEQ ID NO: 33), 5'-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3 '(SEQ ID NO: 34) PCR was carried out using the extracted whole DNA as a template. The resulting PCR products by electrophoresis, it was confirmed that a gene fragment of 1.7 kb size, corresponding to the phaC1 _{Ps6 -19} gene, and to obtain a phaC1 _{Ps6 -19} gene.

phaC1 _Ps6 to _-19 expressing the synthase, pSYL105 vector (Lee et al . , Biotech. Bioeng ., 1994, 44: 1337-1347) in Ralstonia eutropha A pReCAB recombinant vector was prepared by digesting a DNA fragment containing the PHB-producing operon derived from H16 with BamHI / EcoRI and inserting it into the BamHI / EcoRI recognition site of pBluescript II (Stratagene Co., USA). The pReCAB vector contains the PHA synthase (phaC _RE ) and the monomeric donor enzyme (phaA _RE And phaB _RE ) are constantly expressed by the PHB operon promoter. BstBI / SbfI recognition site was removed BstBI each position and inherent priority to make phaC1 _{Ps6 -19} synthase gene fragments, one containing only the ends without changing the amino acid SDM (site directed mutagenesis) methods, BstBI / SbfI recognition (SEQ ID NO: 35), 5'-CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTC TC -3 '(SEQ ID NO: 36), 5'- (SEQ ID NO: 37), 5'-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC -3 '(SEQ ID NO: 38), 5'-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAA CG 3' Overlapping PCR was performed using GTA CGT GCA CGA ACG GTG ACG CTG GCA TGA GTG -3 '(SEQ ID NO: 39), 5'-aac ggg agg gaa cct gca gg -3' (SEQ ID NO: 40). The pReCAB vector was digested with BstBI / SbfI to obtain R. eutropha H16, remove the PHA synthase (phaC _RE) and then by inserting the phaC1 _{Ps6 -19} gene obtained above in the BstBI / SbfI recognition site to prepare a recombinant vector pPs619C1-ReAB.

3 amino acid positions affecting the activity of short chain length (SCL) were found through amino acid sequence analysis, and primers [5'-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACC ACC- - GGT GGT ATC AAG CAC GGT CAC CAG CAA GGT CAG- 3 '(SEQ ID NO: 42), 5'-CGA GCA GCG GGC ATA TC A TGA GCA TCC TGA ACC CGC- TCA GGA TGC TCA TGA TAT GCC CGC TGC TCG- 3 '(SEQ ID NO: 44), 5'-atc aac ctc atg acc gat gcg atg gcg ccg acc- 3' (SEQ ID NO: 45), 5'- ggt cgg cgc cat cgc atc ggt cat gag gtt gat- 3 ' ( SEQ ID NO: 46) using an SDM method using, E130D, S325T, phaC1 _Ps6 containing Q481M _-19 synthase mutant of phaC1 _Ps6 a pPs619C1300-ReAB containing 300 _-19 .

In order to construct an always expressed system of the operon type in which the propionyl-CoA transferase is expressed in the same manner, propionyl-CoA transferase (CP-PCT) derived from Clostridium propionicum Respectively. CP-PCT was constructed by introducing chromosomal DNA of Clostridium propionikum into a primer [5'-GGAATTCATGAGAAAGGTTCCCATTATTACCGCAGATGA-3 '(SEQ ID NO: 47), 5'-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg- (SEQ ID NO: 48)] (SEQ ID NO: 31). At this time, the NdeI site existing in the original wild-type CP-PCT was removed by SDM method for ease of cloning, and the primer [5'-agg cct gca ggc gga taa caa ttt cac (SEQ ID NO: 49), 5'-gcc cat agt tct aga tta gga ctt cat ttc c-3 '(SEQ ID NO: 50). The pPs619C1300-ReAB vector was digested with SbfI / NdeI to obtain Ralstonia eutrophus The pPs619C1300-CPPCT recombinant vector was prepared by removing the H16-derived monomer-supplying enzyme (phaA _RE and phaB _RE ) and inserting the PCR-cloned CP-PCT gene into the SbfI / NdeI recognition site.

Next, primers [5'-CGCCGGCAGGCCTGCAGG-3 '(SEQ ID NO: 51), 5'-GGCAGGTCAGCCCATATGTC (SEQ ID NO: 51)) were prepared using the above prepared pPs619C1300-CPPCT as a template to introduce a random mutagenesis into the CP- -3 '(SEQ ID NO: 52) was used to perform error-prone PCR under the condition that Mn ^{2 +} was added and the concentration difference of dNTPs was present. Thereafter, PCR was carried out under normal conditions using the above primers to amplify a PCR fragment containing a random mutation. pPs619C1300-CPPCT then by cutting the vector with SbfI / NdeI to remove wild-type CPPCT, to create the ligation mixture was inserted into the amplified PCR fragments to the mutant SbfI / NdeI recognition site introduced in E. coli JM109 ~ 10 ⁵ degree scale Of CP-PCT library. The prepared CP-PCT library was grown in a polymer detection medium (LB agar, glucose 20 g / L, 3HB 1 g / L, Nile red 0.5 μg / ml) for 3 days, Candidates of 80 individuals were selected first. These candidates were subjected to liquid culture (LB agar, glucose 20g / L, 3HB 1g / L, ampicillin 100mg / L, 37 ℃) for 4 days under the conditions of polymer production and analyzed by FACS (Florescence Activated Cell Sorting) CP-PCT Variant 512 (including nucleic acid substitution A1200G) and CP-PCT Variant 522 (including nucleic acid substitutions T78C, T669C, A1125G and T1158C) were selected. Various CP-PCT variants were obtained by random mutagenesis using the above-mentioned error-prone PCR method based on the above-mentioned primary mutants (CP-PCT Variant 512 and CP-PCT Variant 522) CP-PCT Variant 540 (including Val193Ala and silent mutants T78C, T669C, A1125G, and T1158C) was secondarily screened. To amplify the PCR fragment containing the CpPct540 mutation, the above-mentioned primers [5'-agg cct gca ggc gga taa caa ttt cac aca gg- 3 ', 5'-gcc cat atg tct aga tta gga ctt cat ttc c-3 '] under normal conditions. The pPs619C1300-CPPCT vector was digested with SbfI / NdeI to remove the CPPCT fragment, and the amplified CpPct540 PCR fragment was inserted into the SbfI / NdeI recognition site to prepare a ligation mixture to prepare the pPs619C1300-CPPCT540 vector.

Further, the above prepared phaC1 _{Ps6 -19} synthase variant (phaC1 _Ps6-19 300) primers [5'-gaa ttc gtg ctg tcg agc cgc ggg cat atc- 3 '( SEQ ID NO: 53) on the basis of, 5'- derived from Pseudomonas sp. MBEL 6-19 with amino acid sequences mutated E130D, S477F and Q481K using a SDM (site directed mutagenesis) method using gat atg ccc gcg gct cga cag cac gaa ttc- 3 '(SEQ ID NO: 54) A vector pPs619C1310-CPPCT540 containing the PHA synthase mutant (phaC1 _Ps6-19 310) was prepared (Fig. 2).

Next, primers [5'-ATGCCCGGAGCCGGTTCGAA-3 '(SEQ ID NO: 55) and 5'-GAAATTGTTATCCGCCTGCAGG-3' (SEQ ID NO: 55) were prepared using the pPs619C1310-CPPCT540 vector prepared above as a template to introduce a random mutagenesis into the PHA synthase gene. 3 '(SEQ ID NO: 56)] was used to perform error-prone PCR. After performing error-prone PCR, PCR was performed again using the above primers to amplify the PCR fragment containing the mutation, and the amplified mutants were inserted into the BstBI / SbfI site of the pPs619C1310-CPPCT540 vector to construct a library of mutants Respectively. The prepared mutant library was transformed into E. coli XL-1 Blue and cultured in PHB detection medium (LB agar, glucose 20 g / L, Nile red 0.5 μg / ml) for 3 days. The final screened mutants through screening after incubation were pPs619C1249.18H with the mutated amino acid sequence of L18H, V24A, K91R, M128V, E130D, N246S, S325T, S477G, Q481K and A527S. Then, PCR was performed under the general conditions using a primer to amplify the PCR fragment containing pPs619C1249.18H. The pPs619C1310-CPPCT540 vector was digested with BstBI / SbfI to remove the part of pPs619C1310. A ligation mixture in which the PCR fragment of the amplified pPs619C1249.18H was inserted into the BstBI / SbfI recognition site was used to prepare a pPs619C1249.18H-CPPCT540 vector 2).

1-3. 5'- UTR  Sequence-regulated PHA  Production of vectors expressing synthetic enzymes

The UTR portion at the BstBI site in the plasmid (FIG. 2) containing the PHA synthetase mutant prepared in Example 1-2 was amplified by PCR using either one of SEQ ID NO: 1 to SEQ ID NO: 25 prepared in Example 1-1 A series of variant family vectors were substituted for the 5'-UTR mutant sequence. (5'-ggatc ttcgaa ta- (one UTR mutant sequence of SEQ ID NOS: 1 to 25) -atgagtaacaagagtaacgatgagttg-3 '(SEQ ID NO: 1 to 25 as a primer having a mutation sequence, 57) and 5'-ggctcg gcatgc cgccgttgtg-3 '(SEQ ID NO: 58) as R primer. The primer includes a BstBI restriction enzyme sequence in the F primer and an SphI restriction enzyme sequence in the R primer, and the R primer partial sequence is designed to include the SphI restriction enzyme site in the sequence of the PHA synthase. The DNA fragment containing the mutation sequence was amplified by PCR using the above primers for 1 minute at 95 ° C, 35 cycles at 55 ° C for 30 seconds, 72 ° C for 50 seconds, and 72 ° C for 10 minutes, A series of mutant group vectors were prepared by replacing the 5'-UTR portion with the 5'-UTR mutant sequence of SEQ ID NO: 1 to SEQ ID NO: 25 prepared in Example 1-1 using the cloning technique.

1-3. Preparation of Recombinant Escherichia coli and 2- Hydroxybutyrate  Preparation of polymer

The vector prepared above was introduced into Escherichia coli XL1-Blue (Stratagene, USA), and then cultured in MR medium containing 2 g / L 2-hydroxybutyrate and 100 mg / L ampicillin under the conditions of 30 DEG C and 250 rpm for 3 days Lt; / RTI > The composition of the MR medium are as follows (in 1 _{liter) KH 2 PO 4 6.67g, (} NH 4) 2HPO 4 4g, MgSO 4 · 7H 2 O 0.8g, citric acid 0.8g, and trace metal solution 5mL. Here, Trace metal solution is in 1 liter of _{5M HCl 5mL, FeSO 4 · 7H} 2 O 10g, CaCl 2 2g, ZnSO 4 · 7H 2 O 2.2g, MnSO 4 · 4H 2 O 0.5g, CuSO 4 · 5H 2 O, 0.1 g of (NH ₄ ) 6 Mo ₇ O ₂ .4H ₂ O, and 0.02 g of Na ₂ B ₄ O ₂ .10H ₂ O.

Example  2. 5'- UTR  Sequence-regulated pHA  In the transformant strains containing the synthetic enzyme, PHA  Content check

To confirm the concentration and monomer composition of the polymers accumulated in the transformant strain prepared in Example 1, about 30 mg of the dried cell pellet was subjected to methanolysis to convert the monomer components to methyl esters. Benzoic acid was used as an internal standard. The result was performed by the method described in Braunegg et al. (Eur J Appl Microbiol. 6: 29-37, 1978) using an Agilent 6890N GC system (Agilent Technologies, Palo Alto, CA) for gas chromatographic analysis.

The relative expression level (%) and the intracellular PHA content (%) of the PHA enzyme according to the 5'-UTR sequence introduced into the strain are summarized in Table 1 below and the PHA polymer content (Figure 1). ≪ tb > < TABLE >

UTR 13, 14, 16, 17, 18 and 25 were found to increase expression of PHA synthase gene and ultimately to increase intracellular polyhydroxyalkanoate content. However, in the case of UTR 19 and 22, the PHA content was slightly decreased compared to the original sequence, while the expression level of the enzyme increased, whereas the expression level of the UTR 19 and 22 can be applied to improve the polymer content.

In addition, UTRs 1 to 12, 15, 20, 21, 23, and 24 decreased expression of PHA synthase gene and ultimately decreased intracellular polyhydroxyalkanoate content. In particular, the expression levels of UTR 8 and 15 were only 3-4% of the original sequence, but the PHA content was 40-50%.

Mutant UTR sequence SEQ ID NO: Of PHA enzymes
Relative expression level (%) Intracellular PHA content (%) UTR1 CTACCGT CAGAGAGAC ATCTCTCTG One not detected 1.5 UTR2 TTACCGT CAGAGAGAC TTCGCTCTG 2 not detected 1.0 UTR3 CAACCGT CAGAGAGAC ATCTCTTTG 3 0.2 5.2 UTR4 CTACCGT CAGAGAGAC ATATCTCTG 4 not detected 1.2 UTR5 CTACCGT CAGAGAGAC ATTTCTTTG 5 not detected 2.9 UTR6 TTACCTT CAGAGAGAC ATCTCTTTG 6 0.1 2.5 UTR7 CTAACCT CAGAGAGAC ATCTCTTTG 7 0.6 12.5 UTR8 CTACCCT CAGAGAGAC CGCTCTTCG 8 2.8 44.9 UTR9 TTACCCT CAGAGAGAC CCCTTTTTG 9 78.5 58.2 UTR10 CTACCCT CAGAGAGAC ATCTGTTTG 10 31.9 44.5 UTR11 TTCTAGC CAGAGAGAC GTTCTCGTC 11 57.9 50.2 UTR12 GGGCGGT CAGAGAGAC GTTCTCGTC 12 7.0 26.3 UTR13 CCACACG CAGAGAGAC GTGACAATC 13 331.6 60.9 UTR14 TTATTCA CAGAGAGAC ATCCCGGTT 14 216.3 67.2 UTR15 AGTCGGC CAGAGAGAC AATTTGTGA 15 3.8 44.5 UTR16 AAAAGAA CAGAGAGAC AAACATCGG 16 355.1 61.0 UTR17 TAAAGAA CAGAGAGAC AGACACAGG 17 348.6 61.2 UTR18 AGACGAC CAGAGAGAC GAACACCGG 18 150.8 62.4 UTR19 AATCAAA CAGAGAGAC CGACAGTGA 19 133.7 54.3 UTR20 TGCTCCTGCC AAAGGAGCA TCACTC 20 not detected 5.0 UTR21 TGCTCCTGCC AAAGGAGCA TCGCTC 21 not detected 4.3 UTR22 TACTCTTGCC AAAGGAGCA TCACTC 22 181.1 49.2 UTR23 TGCATGGCTG AAAGGAGCA TCAGGC 23 30.0 34.3 UTR24 GCAGGACGCT AAAGGAGCA TCATGC 24 33.3 56.1 UTR25 ACAGGGTGTG AAAGGAGCA TCTTAC 25 602.0 60.2 Original ReC GTGACGG CAGAGAGAC AATCAAATC 26 100.0 59.1

* not detected: no protein band due to Western blot (very low expression level)

<110> LG CHEM, LTD. <120> Method for controlling polyhydroxyalkanoate using sequence          variation in 5'-untranslated region <130> DPP20147707KR <160> 58 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR1 <400> 1 ctaccgtcag agagacatct ctctg 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR2 <400> 2 ttaccgtcag agagacttcg ctctg 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR3 <400> 3 caaccgtcag agagacatct ctttg 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR4 <400> 4 ctaccgtcag agagacatat ctctg 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR5 <400> 5 ctaccgtcag agagacattt ctttg 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR6 <400> 6 ttaccttcag agagacatct ctttg 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR7 <400> 7 ctaacctcag agagacatct ctttg 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR8 <400> 8 ctaccctcag agagaccgct cttcg 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR9 <400> 9 ttaccctcag agagacccct ttttg 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR10 <400> 10 ctaccctcag agagacatct gtttg 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR11 <400> 11 ttctagccag agagacgttc tcgtc 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR12 <400> 12 gggcggtcag agagacgttc tcgtc 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR13 <400> 13 ccacacgcag agagacgtga caatc 25 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR14 <400> 14 ttattcacag agagacatcc cggtt 25 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR15 <400> 15 agtcggccag agagacaatt tgtga 25 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR16 <400> 16 aaaagaacag agagacaaac atcgg 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR17 <400> 17 taaagaacag agagacagac acagg 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR18 <400> 18 agacgaccag agagacgaac accgg 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR19 <400> 19 aatcaaacag agagaccgac agtga 25 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR20 <400> 20 tgctcctgcc aaaggagcat cactc 25 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR21 <400> 21 tgctcctgcc aaaggagcat cgctc 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR22 <400> 22 tactcttgcc aaaggagcat cactc 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR23 <400> 23 tgcatggctg aaaggagcat caggc 25 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR24 <400> 24 gcaggacgct aaaggagcat catgc 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> UTR25 <400> 25 acagggtgtg aaaggagcat cttac 25 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Original ReC <400> 26 gtgacggcag agagacaatc aaatc 25 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> 5'-UTR, wherein n is A, T, G or C <400> 27 nnnnnnncag agagacnnnn nnnnn 25 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> 5'-UTR, wherein n is A, T, G or C <400> 28 nnnnnnnnnn aaaggagcat cnnnn 25 <210> 29 <211> 559 <212> PRT <213> Pseudomonas sp. 6-19 <220> <221> PEPTIDE &Lt; 222 > (1) .. (559) <223> PHA synthase <400> 29 Met Ser Asn Lys Ser Asn Asp Glu Leu Lys Tyr Gln Ala Ser Glu Asn   1 5 10 15 Thr Leu Gly Leu Asn Pro Val Val Gly Leu Arg Gly Lys Asp Leu Leu              20 25 30 Ala Ser Ala Arg Met Val Leu Arg Gln Ala Ile Lys Gln Pro Val His          35 40 45 Ser Val Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Leu      50 55 60 Leu Gly Lys Ser Gly Leu Gln Pro Thr Ser Asp Arg Arg Phe Ala  65 70 75 80 Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr                  85 90 95 Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Asp Glu Ser Asn             100 105 110 Leu Ala Pro Lys Asp Val Ala Arg Gly His Phe Val Ile Asn Leu Met         115 120 125 Thr Glu Ala Met Ala Pro Thr Asn Thr Ala Ala Asn Pro Ala Ala Val     130 135 140 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 145 150 155 160 His Leu Ala Lys Asp Leu Val His Asn Gly Gly Met Pro Ser Gln Val                 165 170 175 Asn Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Val Thr Glu Gly             180 185 190 Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Lys Pro         195 200 205 Thr Thr Glu Gln Val Tyr Glu Arg Pro Leu Leu Val Val Pro Pro Gln     210 215 220 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala 225 230 235 240 Arg Phe Cys Leu Arg Asn Asn Val Gln Thr Phe Ile Val Ser Trp Arg                 245 250 255 Asn Pro Thr Lys Glu Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Glu             260 265 270 Ala Leu Lys Glu Ala Val Asp Val Val Thr Ala Ile Thr Gly Ser Lys         275 280 285 Asp Val Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala     290 295 300 Leu Leu Gly His Tyr Ala Ile Gly Glu Asn Lys Val Asn Ala Leu 305 310 315 320 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu Asp Ser Asp Val Ala                 325 330 335 Leu Phe Val Asn Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser Tyr             340 345 350 Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp         355 360 365 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu     370 375 380 Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 385 390 395 400 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Leu Phe                 405 410 415 Lys Asn Asn Pro Leu Ile Arg Pro Asn Ala Leu Glu Val Cys Gly Thr             420 425 430 Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Phe Ser Leu Ala Gly         435 440 445 Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln     450 455 460 Leu Phe Gly Gly Asn Val Glu Phe Val Leu Ser Ser Ser Gly His Ile 465 470 475 480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr                 485 490 495 Ser Thr Glu Val Ala Glu Asn Ala Asp Glu Trp Gln Ala Asn Ala Thr             500 505 510 Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Gln         515 520 525 Arg Ser Gly Glu Leu Lys Lys Ser Pro Thr Lys Leu Gly Ser Lys Ala     530 535 540 Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg 545 550 555 <210> 30 <211> 1677 <212> DNA <213> Pseudomonas sp. 6-19 <220> <221> gene <222> (1) .. (1677) <223> PHA synthase <400> 30 atgagtaaca agagtaacga tgagttgaag tatcaagcct ctgaaaacac cttggggctt 60 aatcctgtcg ttgggctgcg tggaaaggat ctactggctt ctgctcgaat ggtgcttagg 120 caggccatca agcaaccggt gcacagcgtc aaacatgtcg cgcactttgg tcttgaactc 180 aagaacgtac tgctgggtaa atccgggctg caaccgacca gcgatgaccg tcgcttcgcc 240 gatccggcct ggagccagaa cccgctctat aaacgttatt tgcaaaccta cctggcgtgg 300 cgcaaggaac tccacgactg gatcgatgaa agtaacctcg cccccaagga tgtggcgcgt 360 gggcacttcg tgatcaacct catgaccgaa gcgatggcgc cgaccaacac cgcggccaac 420 ccggcggcag tcaaacgctt ttttgaaacc ggtggcaaaa gcctgctcga cggcctctcg 480 cacctggcca aggatctggt acacaacggc ggcatgccga gccaggtcaa catgggtgca 540 ttcgaggtcg gcaagagcct gggcgtgacc gaaggcgcgg tggtgtttcg caacgatgtg 600 ctggaactga tccagtacaa gccgaccacc gagcaggtat acgaacgccc gctgctggtg 660 gtgccgccgc agatcaacaa gttctacgtt ttcgacctga gcccggacaa gagcctggcg 720 cggttctgcc tgcgcaacaa cgtgcaaacg ttcatcgtca gctggcgaaa tcccaccaag 780 gaacagcgag agtggggcct gtcgacctac atcgaagccc tcaaggaagc ggttgacgtc 840 gttaccgcga tcaccggcag caaagacgtg aacatgctcg gggcctgctc cggcggcatc 900 acttgcactg cgctgctggg ccattacgcg gcgattggcg aaaacaaggt caacgccctg 960 accttgctgg tgagcgtgct tgataccacc ctcgacagcg acgtcgccct gttcgtcaat 1020 gaacagaccc ttgaagccgc caagcgccac tcgtaccagg ccggcgtact ggaaggccgc 1080 gacatggcga aggtcttcgc ctggatgcgc cccaacgatc tgatctggaa ctactgggtc 1140 aacaattacc tgctaggcaa cgaaccgccg gtgttcgaca tcctgttctg gaacaacgac 1200 accacacggt tgcccgcggc gttccacggc gacctgatcg aactgttcaa aaataaccca 1260 ctgattcgcc cgaatgcact ggaagtgtgc ggcaccccca tcgacctcaa gcaggtgacg 1320 gccgacatct tttccctggc cggcaccaac gaccacatca ccccgtggaa gtcctgctac 1380 aagtcggcgc aactgtttgg cggcaacgtt gaattcgtgc tgtcgagcag cgggcatatc 1440 cagagcatcc tgaacccgcc gggcaatccg aaatcgcgct acatgaccag caccgaagtg 1500 gcggaaaatg ccgatgaatg gcaagcgaat gccaccaagc atacagattc ctggtggctg 1560 cactggcagg cctggcaggc ccaacgctcg ggcgagctga aaaagtcccc gacaaaactg 1620 ggcagcaagg cgtatccggc aggtgaagcg gcgccaggca cgtacgtgca cgaacgg 1677 <210> 31 <211> 1575 <212> DNA <213> Clostridium propionicum <220> <221> gene &Lt; 222 > (1) .. (1575) <223> popionyl-CoA transferase <400> 31 atgagaaagg ttcccattat taccgcagat gaggctgcaa agcttattaa agacggtgat 60 acagttacaa caagtggttt cgttggaaat gcaatccctg aggctcttga tagagctgta 120 gaaaaaagat tcttagaaac aggcgaaccc aaaaacatta cctatgttta ttgtggttct 180 caaggtaaca gagacggaag aggtgctgag cactttgctc atgaaggcct tttaaaacgt 240 tacatcgctg gtcactgggc tacagttcct gctttgggta aaatggctat ggaaaataaa 300 atggaagcat ataatgtatc tcagggtgca ttgtgtcatt tgttccgtga tatagcttct 360 cataagccag gcgtatttac aaaggtaggt atcggtactt tcattgaccc cagaaatggc 420 ggcggtaaag taaatgatat taccaaagaa gatattgttg aattggtaga gattaagggt 480 caggaatatt tattctaccc tgcttttcct attcatgtag ctcttattcg tggtacttac 540 gctgatgaaa gcggaaatat cacatttgag aaagaagttg ctcctctgga aggaacttca 600 gtatgccagg ctgttaaaaa cagtggcggt atcgttgtag ttcaggttga aagagtagta 660 aaagctggta ctcttgaccc tcgtcatgta aaagttccag gaatttatgt tgactatgtt 720 gttgttgctg acccagaaga tcatcagcaa tctttagatt gtgaatatga tcctgcatta 780 tcaggcgagc atagaagacc tgaagttgtt ggagaaccac ttcctttgag tgcaaagaaa 840 gttattggtc gtcgtggtgc cattgaatta gaaaaagatg ttgctgtaaa tttaggtgtt 900 ggtgcgcctg aatatgtagc aagtgttgct gatgaagaag gtatcgttga ttttatgact 960 ttaactgctg aaagtggtgc tattggtggt gttcctgctg gtggcgttcg ctttggtgct 1020 tcttataatg cggatgcatt gatcgatcaa ggttatcaat tcgattacta tgatggcggc 1080 ggcttagacc tttgctattt aggcttagct gaatgcgatg aaaaaggcaa tatcaacgtt 1140 tcaagatttg gccctcgtat cgctggttgt ggtggtttca tcaacattac acagaataca 1200 cctaaggtat tcttctgtgg tactttcaca gcaggtggct taaaggttaa aattgaagat 1260 ggcaaggtta ttattgttca agaaggcaag cagaaaaaaat tcttgaaagc tgttgagcag 1320 attacattca atggtgacgt tgcacttgct aataagcaac aagtaactta tattacagaa 1380 agatgcgtat tccttttgaa ggaagatggt ttgcacttat ctgaaattgc acctggtatt 1440 gatttgcaga cacagattct tgacgttatg gattttgcac ctattattga cagagatgca 1500 aacggccaaa tcaaattgat ggacgctgct ttgtttgcag aaggcttaat gggtctgaag 1560 gaaatgaagt cctaa 1575 <210> 32 <211> 524 <212> PRT <213> Clostridium propionicum <220> <221> PEPTIDE &Lt; 222 > (1) .. (524) <223> propionyl-CoA transferase <400> 32 Met Arg Lys Val Pro Ile Ile Thr Ala Asp Glu Ala Ala Lys Leu Ile   1 5 10 15 Lys Asp Gly Asp Thr Val Thr Thr Ser Gly Phe Val Gly Asn Ala Ile              20 25 30 Pro Glu Ala Leu Asp Arg Ala Val Glu Lys Arg Phe Leu Glu Thr Gly          35 40 45 Glu Pro Lys Asn Ile Thr Tyr Val Tyr Cys Gly Ser Gln Gly Asn Arg      50 55 60 Asp Gly Arg Gly Ala Glu His Phe Ala His Glu Gly Leu Leu Lys Arg  65 70 75 80 Tyr Ile Ala Gly His Trp Ala Thr Val Ala Leu Gly Lys Met Ala                  85 90 95 Met Glu Asn Lys Met Glu Ala Tyr Asn Val Ser Gln Gly Ala Leu Cys             100 105 110 His Leu Phe Arg Asp Ile Ala Ser His Lys Pro Gly Val Phe Thr Lys         115 120 125 Val Gly Ile Gly Thr Phe Ile Asp Pro Arg Asn Gly Gly Gly Lys Val     130 135 140 Asn Asp Ile Thr Lys Glu Asp Ile Val Glu Leu Val Glu Ile Lys Gly 145 150 155 160 Gln Glu Tyr Leu Phe Tyr Pro Ala Phe Pro Ile His Val Ala Leu Ile                 165 170 175 Arg Gly Thr Tyr Ala Asp Glu Ser Gly Asn Ile Thr Phe Glu Lys Glu             180 185 190 Val Ala Pro Leu Glu Gly Thr Ser Val Cys Gln Ala Val Lys Asn Ser         195 200 205 Gly Gly Ile Val Val Gly Val Glu Arg Val Val Lys Ala Gly Thr     210 215 220 Leu Asp Pro Arg His Val Lys Val Pro Gly Ile Tyr Val Asp Tyr Val 225 230 235 240 Val Val Ala Asp Pro Glu Asp His Gln Gln Ser Leu Asp Cys Glu Tyr                 245 250 255 Asp Pro Ala Leu Ser Gly Glu His Arg Arg Pro Glu Val Val Gly Glu             260 265 270 Pro Leu Pro Leu Ser Ala Lys Lys Val Ile Gly Arg Arg Gly Ala Ile         275 280 285 Glu Leu Glu Lys Asp Val Ala Val Asn Leu Gly Val Gly Ala Pro Glu     290 295 300 Tyr Val Ala Ser Val Ala Asp Glu Glu Gly Ile Val Asp Phe Met Thr 305 310 315 320 Leu Thr Ala Glu Aly Gly Aly Gly Aly Gly Gly Val Ala Gly Gly Val                 325 330 335 Arg Phe Gly Ala Ser Tyr Asn Ala Asp Ala Leu Ile Asp Gln Gly Tyr             340 345 350 Gln Phe Asp Tyr Tyr Asp Gly Gly Gly Leu Asp Leu Cys Tyr Leu Gly         355 360 365 Leu Ala Glu Cys Asp Glu Lys Gly Asn Ile Asn Val Ser Arg Phe Gly     370 375 380 Pro Arg Ile Ala Gly Cys Gly Gly Phe Ile Asn Ile Thr Gln Asn Thr 385 390 395 400 Pro Lys Val Phe Phe Cys Gly Thr Phe Thr Ala Gly Gly Leu Lys Val                 405 410 415 Lys Ile Glu Asp Gly Lys Val Ile Ile Val Gln Glu Gly Lys Gln Lys             420 425 430 Lys Phe Leu Lys Ala Val Glu Gln Ile Thr Phe Asn Gly Asp Val Ala         435 440 445 Leu Ala Asn Lys Gln Gln Val Thr Tyr Ile Thr Glu Arg Cys Val Phe     450 455 460 Leu Leu Lys Glu Asp Gly Leu His Leu Ser Glu Ile Ala Pro Gly Ile 465 470 475 480 Asp Leu Gln Thr Gln Ile Leu Asp Val Met Asp Phe Ala Pro Ile Ile                 485 490 495 Asp Arg Asp Ala Asn Gly Gln Ile Lys Leu Met Asp Ala Ala Leu Phe             500 505 510 Ala Glu Gly Leu Met Gly Leu Lys Glu Met Lys Ser         515 520 <210> 33 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 atgcccggag ccggttcgaa 20 <210> 36 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 cgttactctt gttactcatg atttgattgt ctctc 35 <210> 37 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 gagagacaat caaatcatga gtaacaagag taacg 35 <210> 38 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 cactcatgca agcgtcaccg ttcgtgcacg tac 33 <210> 39 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 gtacgtgcac gaacggtgac gcttgcatga gtg 33 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 aacgggaggg aacctgcagg 20 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 ctgaccttgc tggtgaccgt gcttgatacc acc 33 <210> 42 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 ggtggtatca agcacggtca ccagcaaggt cag 33 <210> 43 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 cgagcagcgg gcatatcatg agcatcctga acccgc 36 <210> 44 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 gcgggttcag gatgctcatg atatgcccgc tgctcg 36 <210> 45 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 atcaacctca tgaccgatgc gatggcgccg acc 33 <210> 46 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 ggtcggcgcc atcgcatcgg tcatgaggtt gat 33 <210> 47 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 ggaattcatg agaaaggttc ccattattac cgcagatga 39 <210> 48 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 gctctagatt aggacttcat ttccttcaga cccattaagc cttctg 46 <210> 49 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 aggcctgcag gcggataaca atttcacaca gg 32 <210> 50 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 gcccatatgt ctagattagg acttcatttc c 31 <210> 51 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 cgccggcagg cctgcagg 18 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 ggcaggtcag cccatatgtc 20 <210> 53 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 gaattcgtgc tgtcgagccg cgggcatatc 30 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 gatatgcccg cggctcgaca gcacgaattc 30 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 atgcccggag ccggttcgaa 20 <210> 56 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 gaaattgtta tccgcctgca gg 22 <210> 57 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> n is a sequence selected from group consisting of SEQ ID NO.1 to          SEQ ID NO.25 <400> 57 ggatcttcga atanatgagt aacaagagta acgatgagtt g 41 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 ggctcggcat gccgccgttg tg 22

Claims (18)

(5'-untranslated region) comprising the step of culturing a cell comprising a sequence-regulated polyhydroxyalkanoate (PHA) synthetase, the production of a polyhydroxyalkanoate Way.
The method according to claim 1,
Linking the regulated 5'-untranslated region with a PHA synthase gene, and
Introducing into the cell a PHA synthase gene linked to the regulated 5'-non-translated region,
A process for producing polyhydroxyalkanoate.
3. The method of claim 1, wherein the 5'-untranslated region sequence is regulated to a sequence having a free binding energy? G _UTR value defined below as a value less than the? G _UTR value of the wild- Method of preparing eth:
ΔG _UTR = [ΔG _SD + ΔG _start + ΔG _spacing ] - [αΔG _direct + (1-α) ΔG _indirect ]
In the above,? G _SD is the energy released when the Shine-Dalgarno sequence hybridizes with 16S rRNA,
ΔG _start is the energy released when the initiation codon hybridizes with the initiator tRNA (3'-UAC-5 '),
ΔG _spacing is a free energy penalty for an unsmoothed physical distance between the Shine-Dalgano sequence and the initiation codon,
ΔG _direct Is the energy required for the 30S subunit of the ribosome to bind directly to the translation initiation site when the translation initiation site of the transcript is in a temporarily unfolded state,
ΔG _indirect is the energy required for the 30S subunit of the ribosome to bind by indirect force rather than direct binding to the translation initiation site when the translation initiation site of the transcript is in an unfolded state,
α is the population coefficient.
2. The method of claim 1, wherein the regulated 5'-untranslated region comprises the nucleotide sequence of 5'-NNNNNNNNNN-AAAGGAGCATC-NNNN-3 'or 5'-NNNNNNN-CAGAGAGAC-NNNNNNNNN-3' , T, G, or C.
The method of claim 1, wherein the regulated 5'-untranslated region is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: Wherein the nucleotide sequence of the polyhydroxyalkanoate is a nucleotide sequence.
2. The method according to claim 1, wherein the polyhydroxyalkanoate synthase gene is a polyhydroxyalkanoate synthase gene of Pseudomonas sp. Way.
2. The method according to claim 1, wherein the polyhydroxyalkanoate synthase gene
An amino acid sequence of SEQ ID NO: 29; or
A nucleotide sequence corresponding to an amino acid sequence comprising at least one mutation selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 29 , A method for producing polyhydroxyalkanoate.
The method according to claim 1, wherein the polyhydroxyalkanoate synthase gene comprises the amino acid sequence of SEQ ID NO: 29
(i) S325T and Q481M;
(ii) E130D, S325T and Q481M;
(iii) E130D, S325T, S477R and Q481M; And
(iv) a nucleotide sequence corresponding to an amino acid sequence comprising a mutation selected from the group consisting of E130D, S477F and Q481K.
The method of producing a polyhydroxyalkanoate according to claim 1, wherein the cell is a prokaryotic cell.
Comprising the step of adjusting the 5'-untranslated region sequence of a polyhydroxyalkanoate (PHA) synthase,
A method for controlling the content of polyhydroxyalkanoate in a cell.
11. The method of claim 10,
Linking the regulated 5'-untranslated region with a PHA synthase gene, and
Introducing into the cell a PHA synthase gene linked to the regulated 5'-non-translated region,
A method for controlling the content of polyhydroxyalkanoate in a cell.
11. The method of claim 10, wherein the 5'-untranslated region sequences to that of the intracellular poly the free binding energy ΔG _UTR defined value adjusted to the sequence having a value that is less than the value ΔG _UTR of the wild-type sequence, such as hydroxy How to control alkanoate content:
ΔG _UTR = [ΔG _SD + ΔG _start + ΔG _spacing ] - [αΔG _direct + (1-α) ΔG _indirect ]
In the above,? G _SD is the energy released when the Shine-Dalgarno sequence hybridizes with 16S rRNA,
ΔG _start is the energy released when the initiation codon hybridizes with the initiator tRNA (3'-UAC-5 '),
ΔG _spacing is a free energy penalty for an unsmoothed physical distance between the Shine-Dalgano sequence and the initiation codon,
ΔG _direct Is the energy required for the 30S subunit of the ribosome to bind directly to the translation initiation site when the translation initiation site of the transcript is in a temporarily unfolded state,
ΔG _indirect is the energy required for the 30S subunit of the ribosome to bind to the translation initiation site by an indirect force rather than a direct linkage when the translational initiation site of the transcript is in an unfolded state,
α is the population coefficient.
11. The method of claim 10, wherein the regulated 5'-untranslated region comprises the nucleotide sequence of 5'-NNNNNNNNNN-AAAGGAGCATC-NNNN-3 'or 5'-NNNNNNN-CAGAGAGAC-NNNNNNNNN-3' , T, G, or C in a cell.
[Claim 11] The method according to claim 10, wherein the regulated 5'-untranslated region is a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25, wherein the polyhydroxyalkanoate content Lt; / RTI &gt;
The polyhydroxyalkanoate synthase gene according to claim 10, wherein the polyhydroxyalkanoate synthase gene is a polyhydroxyalkanoate synthase gene of Pseudomonas sp. 6-19 (Pseudomonas sp. 6-19) Methods of polymer regulation.
[Claim 11] The method according to claim 10, wherein the polyhydroxyalkanoate synthase gene
An amino acid sequence of SEQ ID NO: 29; or
A nucleotide sequence corresponding to an amino acid sequence comprising at least one mutation selected from the group consisting of E130D, S325T, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 29 , Wherein the content of polyhydroxyalkanoate in the cell is controlled.
11. The method according to claim 10, wherein the polyhydroxyalkanoate synthase gene comprises the amino acid sequence of SEQ ID NO: 29
(i) S325T and Q481M;
(ii) E130D, S325T and Q481M;
(iii) E130D, S325T, S477R and Q481M; And
(iv) a nucleotide sequence corresponding to an amino acid sequence comprising a mutation selected from the group consisting of E130D, S477F and Q481K.
11. The method according to claim 10, wherein the cell is a prokaryotic cell, wherein the content of polyhydroxyalkanoate in the cell is controlled.

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